The overall objective of the proposed research is to investigate the coronary endothelial vasoregulatory function in man. Three specific aims are proposed. Specific Aim #1 is to investigate whether the conflicting reports of acetylcholine-mediated coronary responses in man may be related to the differences in the pathological states of the human coronary arteries studied and/or to inherent differences in regional distribution of muscarinic receptors between the epicardial conduit and intramyocardial resistant arteries. To test these hypotheses, responses to acetylcholine- mediated endothelium-dependent relaxation and -independent contraction will be determined in both epicardial conduit and intramyocardial resistant arteries, with varying degree of intimal proliferation. Regional distribution of muscarinic receptors will also be determined by radioautographic method and receptor subtypes responsible for the contrasting effects will be determined by the functional bioassay methods using selective muscarinic receptor antagonists. Specific Aim #2 is to investigate the cellular mechanisms which may be responsible for the alterations in endothelial vasoregulatory function in human coronary blood vessels and to identify pharmacological interventions which may help to prevent and/or restore coronary endothelial vasoregulatory function. Adult human coronary arteries in our excised heart samples invariably show some degree of intimal proliferation, in contrast to most animal blood vessels. The presence of thickened intimal layer, possibly due to endothelial cell injury and regrowth, may result in an altered biosynthesis and release of EDRF/NO upon stimulation, and may interfere with diffusion and/or degradation of EDRF/NO. To test these hypotheses, it is proposed to investigate the role of: 1) substrate (L- arginine) limitation or depletion for the biosynthesis of EDRF/NO, 2) alterations in the sensitivity of endothelial cell to calcium in the production and/or release of EDRF/NO, 3) alterations in the degradation of EDRF/NO by superoxide anions and the responses of their degradated product, peroxynitrite, and 4) alterations in vascular smooth muscle cell responsiveness to EDRF/NO in human coronary arteries with varying degree of intimal proliferative lesions. Specific Aim #3 is to utilize the newly developed human in vitro intramyocardial resistant artery preparation to investigate the mechanisms of endothelial vasoregulatory function in man, and its alterations in different pathological states. The unique anatomical location of these intramyocardial resistant arteries and their role in the regulation of coronary circulation and blood flow distribution epitomize the importance of a thorough understanding of endothelial vasoregulatory function in man. It is believed that results of the present proposed research will provide important new information on the regulation of myocardial blood flow and the genesis of various cardiovascular and coronary disorders in man.